1. Field of the Invention
The present invention relates to a process for the production of dihomo-xcex3-linolenic acid (DGLA) and a lipid containing DGLA by fermentation using a microorganism capable of producing arachidonic acid (ARA) and having reduced or lost xcex945 desaturation activity.
2. Related Art
DGLA, also known as 8,11,14-eicosatrienoic acid, is known to be present as a constituent fatty acid of fish oils and seaweeds. However, because of the low content of DGLA in fish oils and the like, a purified DGLA product obtained from fish oils and seaweeds is expensive. As relatively efficient production processes, there are fermentation methods using a microorganism capable of producing DGLA (Japanese Unexamined Patent Publication No. 63-14696), methods providing enhanced productivity by adding an additive, such as an unsaturated fatty acid (Japanese Unexamined Patent Publication Nos. 64-47384, and 64-47385), and methods using sesame oil, one of various plant extracts, sesamin, episesamin or the like (Japanese Unexamined Patent Publication Nos. 1-243992, 2-268690, 3-72892 and 3-49688).
Various studies have been carried out regarding the actions of essential fatty acids on an organism, and it is known that in many cases eicosanoids derived from DGLA and ARA are antagonistic. Although it is known that a group of prostaglandin 1 group derived from DGLA exhibit platelet anti-coagulation action, vasodilation activity, bronchdilation activity anti-inflammatory action, and the like, for DGLA orally taken as a fat or oil in foods so as to exhibit the above-mentioned actions, DGLA-containing oil or fat product that has a low content of ARA antagonistic to DGLA is most preferable. Moreover, in the case wherein DGLA is purified from a DGLA-containing fat or oil, a DGLA-containing fat or oil having a low content of ARA is preferable as a starting material. Thus, the development of DGLA-containing fat or oil having a low content of ARA is in urgent demand, but a process for the production of such a fat or oil is not known.
Accordingly, the present invention provides a process for the production of DGLA and a lipid containing DGLA in a simple and efficient manner using a conventional inexpensive medium, and a process for the production of a lipid containing DGLA having a low content of ARA by adding an additive such as a xcex945 desaturase inhibitor.
The present invention have found that DGLA, which is a precursor of ARA, accumulates in a large amount when a microorganism capable of producing ARA and having reduced or lost xcex945 desaturation activity is cultured in a conventional medium, and that a ratio of ARA is further decreased and a ratio of DGLA is further increased when said microorganism is cultured in a medium supplemented with a xcex945 desaturation inhibitor.
Accordingly, the present invention provides a process for the production of DGLA comprising the steps of:
culturing a microorganism having an ability to produce ARA and having reduced or lost xcex945 desaturation activity in a medium to produce DGLA or a lipid containing DGLA; and
recovering the DGLA.
The present invention also provides a process for the production of a lipid containing DGLA comprising the steps of:
culturing a microorganism having an ability to produce ARA and having reduced or lost xcex945 desaturation activity in a medium to produce a lipid containing DGLA; and
recovering the lipid containing DGLA.
The present invention further provides a process for the production of DGLA comprising the steps of:
culturing a microorganism having an ability to produce ARA and having reduced or lost xcex945 desaturation activity in a medium supplemented with a xcex945 desaturase inhibitor, or adding a xcex945 desaturase inhibitor into a medium in which said microorganism has been cultured and further culturing the microorganism to produce DGLA or a lipid containing DGLA; and
recovering the DGLA.
The present invention further provides a process for the production of a lipid containing DGLA comprising the steps of:
culturing a microorganism having an ability to produce ARA and having reduced or lost xcex945 desaturation activity in a medium supplemented with a xcex945 desaturase inhibitor, or adding a xcex945 desaturase inhibitor into a medium in which said microorganism has been cultured and further culturing the microorganism to produce a lipid containing DGLA; and
recovering the lipid containing DGLA.
The present invention moreover provides a process for the production of DGLA comprising the steps of:
culturing a microorganism having an ability to produce ARA and having reduced or lost xcex945 desaturation activity in a medium supplemented with at least one additive selected from the group consisting of sesame oil, peanut oil, an extract obtained by extracting sesame oil with an organic solvent substantially immiscible with sesame oil, an extract of sesame seeds, an extract of Gokahi derived from a medicinal plant, which is Acanthopanax gracilistylus W. W. Smith, Acanthopanax senticosus Harms, Acanthopanax henryi, or Acanthopanax verticillatus Hoo, an extract of Touboku derived from a medicinal plant, an which is Paulownia fortunei Hemsl; or Paulownia tomentosa Steud., an extract of Hakukajihi derived from a medicinal plant, an which is of Ginkgo biloba L., an extract of Hihatsu derived from a medicinal plant, an extract of Piper longum L., which is Saishin (Asiasari radix) derived from medicinal plant, a which is Asiasarum heterotropoides var. mandshuricum, an extract of Asarum sieboldii Miq., an extract of tarragon, an extract of dill seeds, an extract of parsley, an extract of turmeric and an extract of nutmeg, or adding said additive into a medium in which said microorganism has been cultured and further culturing the microorganism to produce DGLA or a lipid containing DGLA, and
recovering the DGLA.
The present invention further provides a process for the production of a lipid containing DGLA comprising the steps of:
culturing a microorganism having an ability to produce ARA and having reduced or lost xcex945 desaturation activity in a medium supplemented with at least one additive selected from the group consisting of sesame oil, peanut oil, an extract obtained by extracting sesame oil with an organic solvent substantially immiscible with sesame oil, an extract of sesame seeds, an extract of Gokahi derived from a medicinal plant, an extract of Acanthopanax gracilistylus W. W. Smith, an extract of Acanthopanax senticosus Harms, an extract of Acanthopanax henryi, an extract of Acanthopanax verticillatus Hoo, an extract of Touboku derived from a medicinal plant, an extract of Paulownia fortunei Hemsl; an extract of Paulownia tomentosa Steud., an extract of Hakukajihi derived from a medicinal plant, an extract of Ginkgo biloba L., an extract of Hihatsu derived from a medicinal plant, an extract of Piper longum L., an extract of Saishin (Asiasari radix) derived from medicinal plant, an extract of Asiasarum heterotropoides var. mandshuricum, an extract of Asarum sieboldii Miq., an extract of tarragon, an extract of dill seeds, an extract of parsley, an extract of turmeric and an extract of nutmeg, or adding said additive into a medium in which said microorganism has been cultured and further culturing the microorganism to produce a lipid containing DGLA, and
recovering the lipid containing DGLA.
In the present invention any microorganisms having an ability to produce ARA and having reduced or lost xcex945 desaturase activity can be used. Microorganism having an ability to produce ARA include those belonging to the genus Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, or Entomophthora. As microorganisms belonging to the genus Mortierella, there are mentioned microorganisms belonging to the subgenus Mortierella, such as Mortierella elongata, Mortierella exiqua, Mortierella hyprophila, Mortierella alpina, and the like. Microorganism used in the present invention, having an ability to produce ARA and having reduced or lost xcex945 desaturase activity can be obtained by mutating the microorganisms having an ability to produce ARA.
For mutagenesis, irradiation of a microorganism with a mutagen, such as radiation (X-ray, xcex3-ray, neutron or ultraviolet light), high temperature treatment, and chemical mutagens may be used. In a mutagenizing procedure, microbial cells are suspended to an appropriate buffer, and a mutagen is added therein. The treated suspension is incubated for an appropriate time, diluted and plated on a solid medium such as agar medium to form colonies of mutated microorganisms.
As chemical mutagens, alkylating agents such as nitrogen mustard, methyl methanesulfonate (MMS), N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG); base analogs such as 5-bromouracil; antibiotics such as mitomycin C; base synthesis inhibitor such as 6-mercaptopurine; pigments such as proflavine; a certain carcinogens such as 4-nitroquinoline-N-oxide; and others such as manganese chloride, potassium permanganese, nitrous acid, hydrazine, hydroxylamine, formaldehyde, nitrofurane compounds may be mentioned. Microorganisms to be treated with a mutagen can be vegetative cells such as mycelium or spores.
As a mutant belonging to the genus Mortierella, Mortierella alpina SAM 1860 (FERM BP-3589) can be preferably used.
For culturing a mutant used in the present invention, spores, mycelium or a previously cultured precultur is inoculated to a liquid medium or solid medium. A liquid medium contains, as a carbon source, glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, and the like, alone or in combination.
As a nitrogen source, an organic nitrogen source such as peptone, yeast extract, malt extract, meat extract, casamino acids, corn steep liquor or urea, and an inorganic nitrogen source such as sodium nitrate, ammonium nitrate, ammonium sulfate or the like can be used alone or in combination. In addition, if necessary, inorganic salts such as phosphates, magnesium sulfate, ferric or ferrous sulfate, cupric sulfate or the like, and minor nutrient components such as vitamins may be used.
The concentration of components in a culture medium should be such that it does not inhibit the growth of microorganisms. Generally and practically, a concentration of carbon source is 0.1 to 30% by weight, preferably 1 to 10% by weight, and a concentration of nitrogen source is 0.01 to 5% by weight, and preferably 0.1 to 2% by weight. Temperature for culturing is 5 to 40xc2x0 C., and preferably 20 to 30xc2x0 C.; and a pH value of a medium is 4 to 10, and preferably 6 to 9. Culturing may be aeration/agitation culturing, shaking culture, or stationary culture. Culturing is usually continued for 2 to 10 days.
In the case wherein a microorganism is cultured in a solid medium, the medium comprises wheat bran, rice hulls, rice bran or the like supplemented with water in an amount of 50 to 100% by weight retuting to the weight of solid materials. Culturing is carried out at 5 to 40xc2x0 C., preferably 20 to 30xc2x0 C. for 3 to 14 days. In this case, the medium can contain nitrogen sources, inorganic salts, and minor nutrient compounds, such as those described above.
According to the present invention, to accelerate an accumulation of DGLA, a substrate of ARA can be added to a medium. As the substrates, hydrocarbons having 12 to 20 carbon atoms such as tetradecane, hexadecane and octadecane, fatty acid having 12 to 20 carbon atoms such as tetradecanoic acid, hexadecanoic acid and octodecanoic acid, a salts thereof, for example, sodium salt or potassium salt, fatty acid esters wherein the fatty acid moiety has 12 to 20 carbon atoms, for example, lower alkyl ester, for example, methyl ester, ethyl ester or propyl ester of such a fatty acid, and a lipid containing such fatty acids as its components, for example, olive oil, soybean oil, cotton seed oil, coconut oil may be mentioned, and they can be used alone or in combination.
Moreover, according to the present invention, to produce a lipid containing DGLA in which a content of ARA is decreased, a producer microorganism is cultured in the presence of a xcex945 desaturase inhibitor, resulting in the accumulation of a large amount of DGLA. In this case, as xcex945 desaturase inhibitors, there are mentioned dioxabicyclo[3.3.0]octane derivatives represented by the following formula (I): 
wherein R1, R2, R3, R4, R5 and R6 independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, or R1 and R2, and/or R4 and R5 together form a methylene group or an ethylene group, and n, m and l are 0 or 1;
piperonyl butoxide, curcumin, and compounds represented by the following formula (II): 
wherein R1 represents a lower alkyl group; R2 represents a hydroxyl group, an alkyl group, an alkoxy group, an alkenyl group or an oxyalkyl group wherein in the case that more than one R2 is present, the R2 may be the same or different, and n is an integer of 0 to 5. The lower alkyl group has 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like. The alkyl group, the alkyl moiety in the alkoxy group, or the oxyalkyl group, and the alkenyl group have 12 to 20 carbon atoms. The xcex945 desaturase inhibitors can be used alone or in combination.
As the dioxabicyclo[3.3.0]octane derivative, in the present invention, sesamin, sesaminol, episesamin, episesaminol, sesamolin, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo-[3.3.0]octane, 2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane or 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane, for example, can be used. These derivatives can be used alone or in the form of a mixture of two or more thereof. Both the optically active form and racemic form can be used.
The dioxabicyclo[3.3.0]octane derivative, one of the xcex945 desaturase inhibitors of the present invention can be obtained by the following procedure. First, an extract composed mainly of the dioxabicyclo[3.3.0]octane derivatives can be obtained from sesame oil according to a method comprising extracting sesame oil with an organic solvent substantially immiscible with sesame oil and capable of extracting and dissolving the compound of the present invention, and concentrating the extract. As the organic solvent, there can be mentioned, for example, acetone, methylethylketone, diethylketone, methanol and ethanol. For example, an extract composed mainly of the compound of the present invention can be obtained by mixing sesame oil homogeneously with an organic solvent as mentioned above, allowing the mixture to stand at a low temperature, carrying out a phase separation according to a customary process, and removing the solvent from the solvent fraction by evaporation.
More specifically, sesame oil is dissolved in 2 to 10 volumes, preferably 6 to 8 volumes of acetone, and the solution is allowed to stand at xe2x88x9280xc2x0 C. overnight. As a result, the oil component is precipitated, and the organic solvent is removed from the obtained filtrate by distillation, whereby an extract composed mainly of the compound of the present invention is obtained. Alternatively, sesame oil is mixed with hot methanol or hot ethanol, the mixture is allowed to stand at room temperature, and the solvent is removed from the solvent fraction to obtain an extract composed mainly of the compound of the present invention. More specifically, sesame oil is mixed with hot methanol (higher than 50xc2x0 C.) or hot ethanol (higher than 50xc2x0 C.) in a volume 2 to 10 times, preferably 5 to 7 times, as large as the volume of the sesame oil to effect a violent extraction. The phase separation is effected by a phase separation when standing at room temperature or a centrifugal separation according to customary procedures, and the solvent is removed from the solvent fraction by distillation to obtain an extract composed mainly of the compound used in the present invention. Furthermore, the supercritical gas extraction can be utilized.
The compound of the present invention can be obtained from an extract as mentioned above by treating the extract by a customary method such as column chromatography, high performance liquid chromatography, recrystallization, distillation, or liquidxe2x80x94liquid countercurrent distribution chromatography. More specifically, by using a reversed phase column (5C18) and methanol/water (60/40) as the eluent, the extract is subjected to high performance liquid chromatography, the solvent is removed by distillation, and the obtained crystal is recrystallized from ethanol to obtain the compound used in the present invention, such as sesamin, episesamin, sesaminol or episesaminol. The sesame oil used in the present invention can be either a purified product or a crude product. Furthermore, sesame seeds or sesame lees (defatted sesame seeds having a residual oil content of 8 to 10%) can be used. In this case, sesame seeds or sesame lees are pulverized if necessary, and then subjected to the extraction according to customary procedures using any solvent, for example, a solvent as mentioned above with respect to the extraction from sesame oil. The extraction residue is separated, and the solvent is removed from the extract by evaporation or the like to obtain an extraction product.
The compound used in the present invention, for example, sesamin, sesaminol, episesamin, episesaminol, sesamolin, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, 2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane or 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane, can be obtained from a sesame seed extract, a sesame lee extract or a crude sesame oil extract according to the same procedures as described above. Moreover, the compound used in the present invention can be obtained from a by-product formed in the sesame oil-preparing process.
Note, sesamin obtained from Asaiasari radix exhibits the same effects as those provided by sesame seeds, sesame bran and sesame oil.
The process for the purification of the compound used in the present invention and the process for obtaining the extract are not limited to those mentioned above, and the compound used in the present invention and the extract composed mainly of the compound of the present invention are not limited to those obtained from sesame oil, sesame lees and sesame seeds, but as is apparent to persons with ordinary skill in the art, all natural substances containing the compound used in the present invention can be used. For example, there can be mentioned Gokahi derived from a medicinal plant which is Acanthopanax gracilistylus W. W. Smith, Acanthopanax senticosus Harms, Acanthopanax henryior, Acanthopanax verticillatus Hoo, Touboku derived from a medicinal plant which is Paulownia fortunei Hemslor, Paulownia tomentosa Steud., Hakukajihi derived from a medicinal plant which is Ginkgo biloba L., Hihatsu derived from a medicinal plant which is Piper longum L., Saishin (Asiasari radix) derived from medicinal plant which is Asiasarum heterotropoides var. mandshuricumor Asarum sieboldii Miq.
The following processes can be adopted for the synthesis of the dioxabicyclo[3.3.0]octane derivative.
For example, sesamin and episesamin can be synthesized according to the process of Beroza et al. [J. Am. Chem. Soc., 78, 1242 (1956)]. Pinoresinol [in the general formula (I), R1 and R4 represent H, R2 and R5 represent CH3, and n, m and l are zero] can be synthesized according to the process of Freundenberg et al. [Chem. Ber., 86, 1157 (1953)]. Furthermore, syringaresinol [in the general formula (I), R1 and R4 represent H, R2, R3, R5 and R6 represent CH3, n is zero, and each of m and l is 1] can be synthesized according to the process of Freundenberg et al. [Chem. Ber., 88, 16 (1955)].
The compound used in the present invention also can be used in the form of a glycoside, to accelerate absorption as far as the glycoside has a specific xcex945 desaturase inhibitory activity.
As embodiments of the compound represented by the formula (II), anisole, methoxyphenol, dimethoxybenzene, diethoxybenzene, trimethoxybenzene, methoxytoluene, 3(2)-tert-butyl-4-hydroxanisole (BHA), eugenol, and the like can be mentioned.
Moreover, as additives added to a culture medium to increase an accumulation of DGLA, sesame oil, peanut oil, an extract obtained by extracting sesame oil with an organic solvent substantially immiscible in sesame oil, an extract of sesame seeds, an extract of Gokahi derived from a medicinal plant, an extract of Acanthopanax gracilistylus W. W. Smith, an extract of Acanthopanax senticosus Harms, an extract of Acanthopanax henryi, an extract of Acanthopanax verticillatus Hoo, an extract of Touboku derived from a medicinal plant, an extract of Paulownia fortunei Hemsl; an extract of Paulownia tomentosa Steud., an extract of Hakukajihi derived from a medicinal plant, an extract of Ginkgo biloba L., an extract of Hihatsu derived from a medicinal plant, an extract of Piper longum L., an extract of Saishin (Asiasari radix) derived from medicinal plant, an extract of Asiasarum heterotropoides var. mandshuricum, an extract of Asarum sieboldii Miq., as well as extracts of spicy plants, such as an extract of tarragon, an extract of dill seeds, an extract of parsley, an extract of turmeric, an extract of nutmeg and the like can be used. These extracts can be prepared using a solvent such as dichloromethane, ethanol, methanol, ethyl ether or the like.
An amount of the above-mentioned additives to be added to a culture medium is as follow. An amount of sesame oil or peanut oil or a total amount of them is 0.001 to 10% by weight per medium, and preferably 0.5 to 10% by weight per medium. An amount of a sesame oil extract and other extract to be added is 3xc3x9710xe2x88x923 to 3xc3x9710xe2x88x921% by weight per medium. An amount of a dioxabicyclo[3.3.0]octane derivatives such as sesamin, sesaminol, episesamin, episesaminol and the like, or a total amount of a combination thereof is 1xc3x9710xe2x88x923 to 1xc3x9710xe2x88x921% by weight per medium.
The additive can be added prior to the inoculation of a producer microorganism or immediately after the inoculation. Alternatively, the additive can be added, after the culturing has started, to a culture medium in which the microorganism is growing or has been grown, followed by further culturing. Moreover, the additive can be added both prior to culturing and during culturing after the culturing has started. In the case wherein the additive is added during culturing, the additive can be added once or more than one time, or continuously.
During the culturing, a large amount of lipid containing DGLA is intracellularly accumulated. In the case wherein a liquid medium is used, DGLA is then recovered by a procedure, for example, described in the following.
After the culturing, the cultured cells are recovered by a conventional solid liquid separation means, such as centrifugation or filtering. The cells are thoroughly washed with water, and preferably dried. The drying can be carried out by lyophilization or air drying. The dried cells are extracted with an organic solvent, preferably in a nitrogen gas flow. As an organic solvent, an ether such as ethyl ether, hexane, a lower alcohol such as methanol or ethanol, chloroform, dichloromethane, petroleum ether, or the like can be used. Moreover, an alternating extraction with methanol and petroleum ether, or an extraction with one phase solvent of chloroform-methanol-water can be successfully used. The solvent is distilled off from the extract under reduced pressure to obtain a lipid containing DGLA in a high concentration.
Alternatively, wet cells can be extracted with a solvent miscible with water, such as methanol or ethanol, or a mixed solvent miscible with water, comprising said solvent and water and/or other solvent. Other procedures are the same as described above for dried cells.
The lipid thus obtained contains DGLA as a component of the lipid such as fat. Although DGLA can be directly isolated, preferably it is isolated as an ester with a lower alcohol, for example, as methyl dihomo-xcex3-linolenate. The esterification accelerates the separation of the target fatty acid from other lipid components, and from other fatty acids produced during the culturing, such as palmitic acid, oleic acid and linoleic acid (these fatty acids are also esterified simultaneously with the esterification of DGLA). For example, to obtain methyl ester of DGLA, the above-mentioned extract is treated with anhydrous methanol/HCl 5 to 10%, or BF3/methanol 10 to 50% at room temperature for 1 to 24 hours.
Methyl ester of DGLA is recovered preferably by extracting the above-mentioned treated solution with an organic solvent such as hexane, an ether such as ethyl ether, or an ester such as ethyl acetate. Next, the resulting extract is dried on, for example, anhydrous sodium sulfate, and the solvent is distilled off preferably under reduced pressure to obtain a mixture comprising fatty acid esters. This mixture contains, in addition to the desired fatty acid HGLA methyl ester, other fatty acid methyl esters, such as methyl parmitate, methyl stearate, methyl oleate and the like. To isolate methyl ester of DGLA from the mixture of these fatty acid methyl esters, column chromatography, low temperature crystallization, the urea-inclusion method, the liquid/liquid countercurrent chromatography method, and the like can be used alone or in combination.
To obtain DGLA from the methyl ester of DGLA, the latter is hydrolyzed with an alkali and freed DGLA is then extracted with an organic solvent, for example, an ether such as an ethyl ether, an ester such as ethyl acetate, or the like.
Moreover, to recover DGLA without using methyl ester, the above-mentioned extracted lipid is subjected to an alkalysis (for example, with 5% sodium hydroxide at room temperature for 2 to 3 hours), and the alkali hydrolysate is extracted and the desired fatty acid DGLA is purified according to a conventional procedure.